MKL-1 and MS-1 cells were grown in RPMI media (Gibco #31800022) and U2OS cells were grown in McCoy’s 5Amedia (Himedia #AT179) supplemented with 10% fetal bovine serum (Gibco #16000-044), 1% GlutaMax (Gibco #35050-061), 1% penstrep (Gibco #15140-122) at 37°C in humidified air incubator containing 5% CO2.
Dual Gene Reporter Assay
U2OS cells were seeded in 6-well plate. After 24 h of seeding, cells were co-transfected with pGL4 NCCR (containing Firefly luciferase gene), pRL.TK (containing Renilla luciferase gene) and plasmids expressing MCV LT and BRD4 proteins using PEI transfection protocol (as described below). After 48 h of post-transfection, Dual-Luciferase reporter assay was performed using manufacturer’s protocol (Promega #E1901). Experiments were repeated independently 3 times with 2 technical repeats each time. The graphs were drawn using GraphPad Prism and statistical significance was calculated using one way ANOVA.
Cells were seeded in an appropriate dish (6-well dish for Luciferase assay) with 50–60% confluency. After 24 h of seeding, media was changed and solution A containing PBS and DNA was prepared. After that solution B containing 1 part of 10 mM PEI (Polyethyleneimine) mixed with 5 parts of PBS, was added dropwise to solution A while vortexing, followed by incubation for 5 min at room temperature. Transfection mixture was added dropwise to the desired plate. Cells were harvested after 48 h of transfection for further analysis. For 6-well plate- Solution A was = 2 mg of DNA + 36 ml of PBS and Solution B = 43.2 ml of PEI-PBS solution.
Transfected cells were lysed in passive lysis buffer from Dual-luciferase kit (#E1901). Lysates were then electrophoresed in 8% and 12% SDS-polyacrylamide gels, transferred to Polyvinylidine fluoride (PVDF) Membrane (Bio-Rad- #1620177) and reacted with Ab5 (1:2000) and BRD4 (Bethyl Laboratories #A301-985A100) (1:2000) overnight at 4°C followed by 1 h incubation at room temperature with anti-mouse antibody (1:5000) Ab5 antibody and Anti- rabbit antibody (1:5000). Detection of peroxidase activity was performed by western lightening plus-ECL reagent (GE Healthcare #RPN2236) and images analyzed in ImageQuant LAS4000. Ab3 and Ab5 were kind gifts of Dr. James Decaprio, Dana Farber Cancer Institute, Boston.
Protein concentration was quantified using Bicinchoninic assay (BCA) in accordance with the manufacturer’s protocol (Pierce BCA Protein assay kit, Thermo Fisher #23225).
For immunoprecipitation assays, cells were pelleted and resuspended in Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM dithiothreitol (DTT) supplemented with protease inhibitors (Complete Roche Applied Science, #5892953001). The resuspended cells were incubated on ice for 10 min, and NP-40 was added to a final concentration of 0.6%. After vortexing and centrifugation at 5,000 rpm for 5 min, the nuclear pellet was resuspended in ice-cold EBC buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5 mM EDTA, β-mercaptoethanol-1:10,000, 0.5% NP-40) supplemented with protease inhibitors) for left for 20 min on ice followed by centrifugation at 14,000 rpm for 10 min. Nuclear proteins were then mixed with 20 µl of protein G Dynabeads(Invitrogen #10004D) (washed thrice with EBC buffer beforehand) along with either 1 µg BRD4 antibody (Bethyl Laboratories #A301-985A100) and 1 µg CM2B4 (Santa Cruz #sc-136172), Ab3 and Ab5 (against MCV T antigens) followed by rotation overnight at 4°C. After overnight incubation, the bound proteins were eluted using laemli sample buffer followed by heating at 99°C for 5 min. These samples were then immunoblotted on 4–12% gradient gel (Biorad #4561094).
For Immunofluoroscence staining, MKL-1 cells were brought to single cell suspension by treating them with 2 mM EDTA for 10 min with gentle pipetting in between. The EDTA containing cell suspension was then diluted in a 1:5 ratio with RPMI + 10% FBS media (1 ml of EDTA contacting cell suspension + 5 ml of RPMI + 10% FBS media). The cells were then adhered on poly-L-Lysine (0.1 mg/ml, diluted in borate buffer) coated coverslips for 2 h, washed once with 1X PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Further they were permeabilized with phosphate buffered saline (PBS) containing 0.5% TritonX-100 for 10 min at room temperature. After permeabilization, cells were blocked with 10% donkey serum for 1 h at room temperature and stained with BRD4 rabbit polyclonal antibody (1:500 dilution, Bethyl Laboratories) and CM2B4 mouse monoclonal Antibody (1:100 dilution, Santa Cruz) overnight at 4°C. Cells were then washed thrice with phosphate buffered saline (PBS) with 0.5% TritonX-100 in the interval of 5 min and then stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit (Invitrogen, A21206), 1:500 dilution and Alexa Fluor 647-conjugated anti-mouse (Invitrogen, A31571), 1:100 dilution) for 1 h at room temperature. Stained cells were mounted in aqueous medium containing DAPI (VectorLaboratories #H-1200) and analyzed and imaged using FV1000 confocal microscope (Olympus) at 60X. Colocalization analysis was done on images of MKL1 cells stained with both BRD4 and MCV LT antibodies using the JaCoP plugin in ImageJ software.